The broad objectives of the proposed research are to identify the molecular mechanisms underlying the function and bioregulation of the Na+K+ATPase isoenzyme, and to develop an understanding of their roles in renal physiology. The Na+K+ATPase plays a critical role in an array of essential functions in all cells, and its contributes directly to the maintenance of normal excitability of the nervous system and heart, and to the normal renal excretion of solutes and water. The first goal will be to characterize the functional properties, subunit assembly relationships, and membrane targeting of each isoenzyme. Using gene transfer methods, cDNAs encoding the major alpha and beta subunit isoforms of the avian Na+K+ATPase will be expressed in tissue cultured mammalian cells, and the biochemical antibodies will be used to study the efficiency of assembly of the different alpha and beta isoforms pairs expressed in these cells, and to investigate the delivery of transfected Na+ K+ATPase molecules to membrane domains of polarized MDCK cells. Chimeric genes and substitution mutants of the isoforms will be constructed and expressed in tissue cultured cells to identify regions of the molecules contributing to distinctive functions. A second major objective will be to analyze the biosynthesis, subunit assembly, and intracellular transport of the Na+K+ATPase isoenzyme expressed in cultured glomerular mesangial cells from the rat. This cell type expresses multiple isoenzyme under basal conditions, and provides a powerful model for dissecting the mechanisms involved in the coordination of Na+K+ATPase isoenzyme expression. Measurements of mRNA and protein abundance for each isoforms will be performed, and the kinetics of gene transcription, translation, and cell surface expression of each isoform will be quantified. Low extracellular concentrations of K+ will be used to induce Na+K+ATPase expression, and the discrete steps involved in the regulatory response of the different isoenzyme will be analyzed. Antisense oligodeoxynucleotides will be used to inhibit synthesis of individual subunit isoforms with the goal of understanding the interplay of the various isoforms within the cell and the control points for regulation of Na+K+ATPase expression. The third major objective will be to examine the expression and regulation of the different isoenzyme along the rat nephron in response to chronic K+ depletion and variations in aldosterone levels. Reverse transcription PCR of RNA isolated from microdissected nephron segments of the rat and in situ hybridization histochemistry of kidney sections will be used to characterize Na+K+ATPase isoform gene expression under basal conditions and in response to these stimuli. These observations will be correlated with immunolocalization studies of kidney sections, and immunoblot analysis and functional Na+K+ATPase assays of microdissected nephron segments. the results of these studies should provide important insights into the molecular basis for the functional diversity and cell type-specific regulation of this multigene family.